Neurons contact each other mostly by synaptic transmission at synapses. The maintenance of synaptic transmission relies on vesicle endocytosis, which recycles fused vesicles for the second round of exocytosis. My goal is to improve our understanding on the cellular and molecular mechanisms underlying synaptic vesicle endocytosis, which are the building block for the maintenance of synaptic transmission and thus the signaling process of the nervous system. Our progress in the last year is described below. 1. Membrane fusion and fission are vital for eukaryotic life. For many decades, it has been proposed that fission is via hemi-fission, which produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fission in live cells. Here we report the observation of a hemi-fused -shaped structure in live neuroendocrine chromaffin cells and pancreatic -cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission. 2. -Synuclein (-syn) missense and multiplication mutations have been suggested to cause neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies. Before causing the progressive neuronal loss, -syn mutations impair exocytosis, which may contribute to eventual neurodegeneration. To understand how -syn mutations impair exocytosis, we developed a mouse model that selectively expressed PD-related human -syn A53T (h--synA53T) mutation at the calyx of Held terminals, where release mechanisms can be dissected with a patch-clamping technique. With capacitance measurement of endocytosis, we reported that h--synA53T, either expressed transgenically or dialyzed in the short term in calyces, inhibited two of the most common forms of endocytosis, the slow and rapid vesicle endocytosis at mammalian central synapses. The expression of h--synA53Tin calyces also inhibited vesicle replenishment to the readily releasable pool. These findings may help to understand how -syn mutations impair neurotransmission before neurodegeneration. 3. The calyx of Held synapse plays an important role in the auditory system, relaying information about sound localization via fast and precise synaptic transmission, which is achieved by its specialized structure and giant size. During development, the calyx of Held undergoes anatomical, morphological, and physiological changes necessary for performing its functions. The large dimensions of the calyx of Held nerve terminal are well suited for direct electrophysiological recording of many presynaptic events that are difficult, if not impossible to record at small conventional synapses. This unique accessibility has been used to investigate presynaptic ion channels, transmitter release, and short-term plasticity, providing invaluable information about basic presynaptic mechanisms of transmission at a central synapse. Here, we review anatomical and physiological specializations of the calyx of Held, summarize recent studies that provide new mechanisms important for calyx development, synaptic transmission, endocytosis, and examine fundamental presynaptic mechanisms, including endocytosis, learned from studies using calyx as a model nerve terminal. 4. Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV. This study is in collaboration with Dr. Muallem. 5. Oxidative stress in the brain is highly prevalent in many neurodegenerative disorders including lysosomal storage disorders, in which neurodegeneration is a devastating manifestation. Despite intense studies, a precise mechanism linking oxidative stress to neuropathology in specific neurodegenerative diseases remains largely unclear. We used molecular, biochemical, immunohistological, and electrophysiological analyses of brain tissues of Cln1 (-/-) mice to study the role(s) of oxidative stress in mediating neuropathology. Our results show that in Cln1 (-/-) mice oxidative stress in the brain via upregulation of the transcription factor, CCAAT/enhancer-binding protein-, stimulated expression of serpina1, which is an inhibitor of a serine protease, neurotrypsin. Moreover, in the Cln1 (-/-) mice, suppression of neurotrypsin activity by serpina1 inhibited the cleavage of agrin (a large proteoglycan), which substantially reduced the production of agrin-22, essential for synaptic homeostasis. Direct whole-cell recordings at the nerve terminals of Cln1 (-/-) mice showed inhibition of Ca(2+) currents attesting to synaptic dysfunction. Treatment of these mice with a thioesterase-mimetic small molecule, N-tert (Butyl) hydroxylamine (NtBuHA), increased agrin-22 levels. Our findings provide insight into a novel pathway linking oxidative stress with synaptic pathology in Cln1 (-/-) mice and suggest that NtBuHA, which increased agrin-22 levels, may ameliorate synaptic dysfunction in this devastating neurodegenerative disease. This study is in collaboration with Dr. Mukherjee.